ABSTRACT Hepatocellular cancer (HCC) is the sixth most common cancer worldwide, and is highly prevalent in low- and middle-income countries (LMIC). China alone accounts for >50% of HCC diagnoses. Surgical removal is the primary and only curative option, yet only 30-40% of newly diagnosed patients are candidates for such treatment. This is because the development of HCC is usually silent and diagnosis occurs relatively late, when the disease has either metastasized or grown too large for surgical removal. Therefore, early diagnosis is the key to achieving positive clinical outcomes, as removal at early-stage ?when the tumor is small and not metastatic? would result in a marked increase in cures in affected patients. Motivated by this hypothesis, the objective of this proposal is to develop a blood-based point-of-care test (POCT) for hepatocellular cancer (HCC). The POCT we have developed, the ?D4 POCT?, is ideally suited for blood-based testing in LMIC settings. The D4 POCT uses polymer brush-coated antibody (Ab) microarrays that have virtually no background binding, yields quantitative results, and achieves picomolar sensitivity within 30 minutes. All reagents are inkjet-printed and stored on D4 POCT cassettes, which do not require refrigeration. Upon direct application of fingerstick blood onto a cassette, analyte capture and detection occur automatically, generating a quantifiable signal obtained by placing the cassette in a small device that magnetically attaches to a smart phone, which images and analyzes microarrays via on-board App. We will develop a multiplexed D4 POCT capable of simultaneously detecting alpha-fetoprotein (AFP), dickkopf-1 (DKK-1) and osteopontin (OPN) that will provide high sensitivity and specificity for HCC diagnosis. Our choice of markers is based on recent clinical evidence showing that the simultaneous measurement of AFP, DKK-1, and OPN as a multiplexed panel provides a promising strategy for early and accurate diagnosis of HCC. The innovation of this proposal is the first complete bottom-up redesign in decades of the ?gold standard? clinical diagnostic?the sandwich immunoassay?as a self-contained point-of-care microarray assay that can be imaged and interrogated by a smart phone. This project will be carried out by a consortium of investigators from Duke University, Zhejiang Provincial People's hospital and our industry partner, Immucor, a leading diagnostics company that has acquired the rights to this technology. After initial validation of the D4 POCT at Duke by meeting specific performance milestones, we will demonstrate technical functionality and clinical potential for use in LMIC settings at the Zhejiang Provincial People's hospital in Guangzhou China using patient-derived finger-stick blood samples. The impact of this project is broad: beyond a new, low-cost, and clinically validated technology for the point-of-care diagnosis of HCC, this diagnostic technology can be utilized to target any analyte for which Ab pairs are available, and is hence broadly applicable to all immunoassays used for diagnosis and prognosis for cancer or other diseases.